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A combined computational and experimental approach reveals the structure of a C/EBP?-Spi1 interaction required for IL1B gene transcription.

ABSTRACT: We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1?, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBP?) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBP? leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBP? DBDs, along with the C/EBP? C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBP?-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBP?'s association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBP? to cognate DNA and in transcription of the C/EBP?-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.

SUBMITTER: Pulugulla SH

PROVIDER: S-EPMC6311527 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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A combined computational and experimental approach reveals the structure of a C/EBPβ-Spi1 interaction required for <i>IL1B</i> gene transcription.

Pulugulla Sree H SH Pulugulla Sree H SH Workman Riley R Rutter Nathan W NW Yang Zhiyong Z Adamik Juraj J Lupish Brian B Macar David A DA El Abdouni Samir S Esposito Emilio Xavier EX Galson Deborah L DL Camacho Carlos J CJ Madura Jeffry D JD Auron Philip E PE

The Journal of biological chemistry 20181024 52

We previously reported that transcription of the human <i>IL1B</i> gene, encoding the proinflammatory cytokine interleukin 1β, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPβ) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPβ leucine zipper is critical for its a ...[more]

PMID: 30355733

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Project description:Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001) between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel-based modeling approach offers practical benefits for probing novel insights into the mode of action of investigational compounds, and for the identification of new target selectivities for drug repurposing applications.

Dataset Information

A combined computational and experimental approach reveals the structure of a C/EBP?-Spi1 interaction required for IL1B gene transcription.

Publications

A combined computational and experimental approach reveals the structure of a C/EBPβ-Spi1 interaction required for <i>IL1B</i> gene transcription.

Similar Datasets

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